自噬降低异常磷酸化tau蛋白所致的神经细胞毒性
发布时间：2018-07-16 08:04
【摘要】：目的观察雷帕霉素和饥饿诱导的自噬对表达异常磷酸化tau蛋白的神经细胞形态、tau蛋白聚集和磷酸化tau降解的影响,探讨这两种经典的诱导自噬方式抑制磷酸化tau的细胞毒性,发挥细胞保护作用的可能机制。方法体外培养小鼠神经瘤母细胞株N2a并转染tau真核表达质粒,蛋白磷酸酯酶抑制剂冈田酸(okadaic acid,OA)诱导tau蛋白异常磷酸化,雷帕霉素(rapamycin,Rapa)或Earle's平衡盐溶液(Earle's balanced salts,EBSS)诱导细胞自噬,巴佛洛霉素A1(Bafilomycin A1,Baf A1)抑制自噬,DAB染色观察表达tau细胞的形态变化;激光共聚焦显微镜观察细胞内tau聚集体;TUNEL染色和caspase-3活性检测细胞凋亡;免疫印迹(immunoblot,IB)检测磷酸化tau和细胞自噬水平。结果过表达tau的细胞胞体变圆,突起减少;OA处理后细胞突起进一步减少甚至消失,胞质出现明显tau聚集体,凋亡细胞增加,剪切型caspase-3水平上调;Rapa和EBSS处理后的细胞形态均有一定程度改善,tau聚集体明显减少,细胞凋亡减少,剪切型caspase-3表达降低;而自噬抑制剂Baf A1处理的细胞变圆,皱缩,胞质大量tau聚集体,凋亡细胞明显增加。IB结果显示Rapa明显降低高分子量的磷酸化tau,而EBSS能明显减少低分子量磷酸化tau的水平。结论 Rapa和EBSS诱导的细胞自噬均能抑制磷酸化tau蛋白的细胞毒作用,但其发挥细胞保护作用的机制不同,Rapa诱导自噬倾向于降解磷酸化tau的寡聚体,而EBSS更易于降解低分子量的磷酸化tau蛋白。
[Abstract]:Objective to investigate the effects of rapamycin and starvation induced autophagy on neuronal morphology tau protein aggregation and phosphorylated tau degradation in neurons expressing abnormal phosphorylated tau protein, and to explore the inhibition of cytotoxicity of phosphorylated tau by these two classical autophagy methods. The possible mechanism of cell protection. Methods Mouse neuroblastoma cell line N2a was cultured in vitro and transfected with tau eukaryotic expression plasmid. The protein phosphatase inhibitor okadaic acidine OA induced abnormal phosphorylation of tau protein, and rapamycin Rapa or Earlehs balanced saltsEBSS induced autophagy. Bafilomycin A1 (Bafilomycin A1) inhibited autophagy dab staining to observe the morphological changes of tau cells, and laser confocal microscopy was used to observe the apoptosis of tau aggregates Tunel and caspase-3 activity. Immunoblotting IB was used to detect phosphorylated tau and autophagy. Results after the over-expression of tau, the cell bodies became round, the processes decreased or even disappeared after treatment with OA, and the cytoplasm showed obvious tau aggregates, and the apoptotic cells increased. Both the up-regulation of caspase-3 level in shear type and the treatment with EBSs can improve the cell morphology, decrease apoptosis and decrease the expression of shearing type caspase-3, while the cells treated with autophagy inhibitor Baf A1 become round and shrink, and the apoptosis of the cells is decreased, and the expression of shearing type caspase-3 is decreased, while the cells treated with autophagy inhibitor Baf A1 become round and shrink. The results showed that Rapa significantly decreased the phosphorylation of tau by high molecular weight, while the level of low molecular weight phosphorylated tau was significantly decreased. Conclusion both Rapa and EBSS-induced autophagy can inhibit the cytotoxicity of phosphorylated tau protein, but the mechanisms of its protective effect are different. Rapa induced autophagy tends to degrade the oligomers of phosphorylated tau. EBSS is easier to degrade low molecular weight phosphorylated tau protein.
【作者单位】： 安徽医科大学基础医学院;安徽医科大学生物药物研究所;安徽医科大学药学院;
【基金】：国家自然科学基金资助项目(No 81302755)
【分类号】：R96
，
本文编号：2125771
[Abstract]:Objective to investigate the effects of rapamycin and starvation induced autophagy on neuronal morphology tau protein aggregation and phosphorylated tau degradation in neurons expressing abnormal phosphorylated tau protein, and to explore the inhibition of cytotoxicity of phosphorylated tau by these two classical autophagy methods. The possible mechanism of cell protection. Methods Mouse neuroblastoma cell line N2a was cultured in vitro and transfected with tau eukaryotic expression plasmid. The protein phosphatase inhibitor okadaic acidine OA induced abnormal phosphorylation of tau protein, and rapamycin Rapa or Earlehs balanced saltsEBSS induced autophagy. Bafilomycin A1 (Bafilomycin A1) inhibited autophagy dab staining to observe the morphological changes of tau cells, and laser confocal microscopy was used to observe the apoptosis of tau aggregates Tunel and caspase-3 activity. Immunoblotting IB was used to detect phosphorylated tau and autophagy. Results after the over-expression of tau, the cell bodies became round, the processes decreased or even disappeared after treatment with OA, and the cytoplasm showed obvious tau aggregates, and the apoptotic cells increased. Both the up-regulation of caspase-3 level in shear type and the treatment with EBSs can improve the cell morphology, decrease apoptosis and decrease the expression of shearing type caspase-3, while the cells treated with autophagy inhibitor Baf A1 become round and shrink, and the apoptosis of the cells is decreased, and the expression of shearing type caspase-3 is decreased, while the cells treated with autophagy inhibitor Baf A1 become round and shrink. The results showed that Rapa significantly decreased the phosphorylation of tau by high molecular weight, while the level of low molecular weight phosphorylated tau was significantly decreased. Conclusion both Rapa and EBSS-induced autophagy can inhibit the cytotoxicity of phosphorylated tau protein, but the mechanisms of its protective effect are different. Rapa induced autophagy tends to degrade the oligomers of phosphorylated tau. EBSS is easier to degrade low molecular weight phosphorylated tau protein.
【作者单位】： 安徽医科大学基础医学院;安徽医科大学生物药物研究所;安徽医科大学药学院;
【基金】：国家自然科学基金资助项目(No 81302755)
【分类号】：R96
，
本文编号：2125771
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